Membrane transport proteins are a large class of integral membrane proteins involved in the movement of small molecules across cellular membranes. Many of these proteins, known as secondary active transporters, use pre-existing substrate concentration gradients as an energy source for translocating another substrate against its concentration gradient. Every organism expresses dozens of different secondary transporter proteins with a diverse array of structural folds, and each protein is specific for a different substrate, which range from ions to neurotransmitters. A detailed understanding of the mechanism of each membrane transport protein requires knowledge of its three-dimensional structure in a number of different conformational states, as well as identification of the binding regions for the substrate or substrates. Studies from our group over the last year have provided insights into a number of biomedically important transporters, as detailed below. In 2014, we identified the structural fold of a secondary transporter responsible for sodium-coupled phosphate uptake in the kidney, called NaPi-IIa, by identifying an evolutionary relationship with a known structure of a sodium-coupled dicarboxylate transporter called VcINDY, which was then used as a template for homology modeling. Within that model we had predicted binding sites for two of the three required sodium ions (Na2 and Na3) and for the phosphate group (Fenollar-Ferrer et al, Biophysical Journal, 2014). However, the location of Na1, which is the first sodium ion to bind, was unresolved. We used our structural model, combined with biochemical and electrophysiological measurements from the Werner and Forster laboratories, to develop a detailed prediction for the residues that coordinate this sodium ion (ref. 1). These results provide an important step forward in identifying the molecular origins of sodium stoichiometry differences in NaPi-II homologs. Homology modeling was also used to generate a structural model of a homolog of vesicular monoamine transporters from Bacillobrevis brevis, BbMAT. A protein of unknown function, YajR, was used as a template for the homology modeling of BbMAT, allowing the identification of potentially interesting pathway-lining ionizable residues. Modification of those residues by our collaborators from the Schuldiner and Singh laboratories demonstrated that they play a key role in proton-dependence of transport, and providing important steps forward in understanding the mechanisms of neurotransmitter transport (ref. 2). A similar molecular modeling strategy was useful in identifying the structural location and behavior of glycosylation sites in a mammalian osmolyte transporter, BGT1, driving experimental studies by the Ziegler lab of the role of glycosylation in trafficking and plasma membrane insertion of this protein in the kidney (ref. 3). We have used structural modeling to explore the conformational mechanism of the oligopeptide symporter, PepT1, responsible for proton-driven uptake of peptides and drugs into the intestine, and a member of the largest group of secondary transporters, the major facilitator superfamily (MFS). Our collaborators in the Newstead laboratory have reported structures of PepT homologs from Shewanella oniedensis and Streptococcus thermopiles (PepTSo and PepTSt, respectively), in cytoplasm-facing conformations. Repeat-swap models based on these structures were outward-facing. These structural models were compared with the crystal structures, and with the results of molecular dynamics simulations, spectroscopic analysis, and functional assays carried out by the Newstead/Fowler groups. Together these results were used to derive a molecular mechanism for alternating access for MFS proteins that is more detailed than the simplistic rocker-switch or rocking bundle mechanisms proposed previously (ref. 4). Separately, we have made progress in exploring the diversity of conformational mechanisms in secondary transporters. Specifically, we have examined whether elevator-like conformational mechanisms, as described previously only for the aspartate transporter GltPh, are used by other transporter families with different structural folds. Using an updated - and more accurate - protocol for repeat-swap modeling, we predicted that the concentrative nucleoside transporter homolog from Vibrio cholerae, VcCNT also uses an elevator-type conformational mechanism (ref. 5). Finally, we used a systematic bioinformatic analysis to demonstrate that structures of two apparently unrelated families of transporters, the paminobenzoyl-glutamate transporter (AbgT) and the divalent anion sodium symporter (DASS) families, in fact have the same general architecture in common (ref. 6), providing novel avenues for studying the modes of substrate binding and conformational change in both these protein families.